Erythropoiesis is the production of red blood cells which occurs to offset cell destruction. Erythropoiesis is a controlled physiological mechanism that enables sufficient red blood cells to be available for proper tissue oxygenation. Naturally occurring human erythropoietin (EPO) is produced in the kidney and is the humoral plasma factor which stimulates red blood cell production (Carnot, P and Deflandre, C (1906) C.R. Acad. Sci. 143: 432; Erslev, A J (1953 Blood 8: 349; Reissmann, K R (1950) Blood 5: 372; Jacobson, L O, Goldwasser, E, Freid, W and Plzak, L F (1957) Nature 179: 6331–4). Naturally occurring EPO stimulates the division and differentiation of committed erythroid progenitors in the bone marrow and exerts its biological activity by binding to receptors on erythroid precursors (Krantz, B S (1991) Blood 77: 419).
Erythropoietin has been manufactured biosynthetically using recombinant DNA technology (Egrie, J C, Strickland, T W, Lane, J et al. (1986) Immunobiol. 72: 213–224) and is the product of a cloned human EPO gene inserted into and expressed in the ovarian tissue cells of the Chinese hamster (CHO cells). The primary structure of the predominant, fully processed form of human erythropoietin (hEPO) is illustrated in FIG. 1 of Egrie et al. There are two disulfide bridges; between Cys7–Cys161 and Cys29–Cys33. The molecular weight of the polypeptide chain of EPO without the sugar moieties is 18,236 Da. In the intact EPO molecule, approximately 40% of the molecular weight is accounted for by the carbohydrate groups that glycosylate the protein at glycosylation sites on the protein (Sasaki, H, Bothner, B, Dell, A and Fukuda, M (1987) J. Biol. Chem. 262: 12059).
Because human erythropoietin is essential in red blood cell formation, the hormone is useful in the treatment of blood disorders characterised by low or defective red blood cell production. Clinically, EPO is used in the treatment of anemia in chronic renal failure patients (CRF) (Eschbach, J W, Egri, J C, Downing, M R et al. (1987) NEJM 316: 73–78; Eschbach, J W, Abdulhadi, M H, Browne, J K et al. (1989) Ann. Intern. Med. 111: 992; Egrie, J C, Eschbach, J W, McGuire, T, Adamson, J W (1988) Kidney Intl. 33: 262; Lim, V S, Degowin, R L, Zavala, D et al. (1989) Ann. Intern. Med. 110: 108–114) and in AIDS and cancer patients undergoing chemotherapy (Danna, R P, Rudnick, S A, Abels, R I In: M B, Garnick, ed. Erythropoietin in Clinical Applications-An International Perspective. New York, N.Y.: Marcel Dekker; 1990: p. 301–324). However, the bioavailability of commercially available protein therapeutics such as EPO is limited by their short plasma half-life and susceptibility to protease degradation. Several concepts, including pegylated EPO derivatives, have been proposed to overcome these disadvantages.
Common approaches for preparing pegylated proteins yield mixtures of mono- and oligo-pegylated proteins. Moreover, the polyethylene glycol compound (PEG) is bound at several positions of the proteins depending on the amount and reactivities of the available reactive groups on the protein surface. Such a mixture may have severe shortcomings: PEG may be bound at positions which interact with the protein specific receptor and conclusively reduce or even prohibit therapeutic efficacy. To solve this drawback either separation and purification of active ingredients of such a mixture or a selective synthetic route to avoid formation is required. Avoiding formation of any mixtures would greatly simplify the synthesis of a pure active pharmaceutical ingredient in terms of a single positional isomer in essentially higher yields. This is particularly true because separation of positional isomers of PEG-protein mixtures may not be possible using common production scale-synthesis.
Several methods have been proposed for the selective modification of recombinantly produced polypeptides.
European Patent Application EP 651,761 discloses the selective modification of recombinantly produced polypeptides at terminal α-carbon reactive groups. The first step in the method is to form a recombinantly produced polypeptide so that it is protected at the terminal α-carbon reactive group with a biologically added protecting group. The biologically added protecting group is preferably an amino acid, peptide and/or polypeptide that contains at least one site that is cleavable enzymatically or chemically, and preferably has a sequence that is not present in the sequence of the desired polypeptide. Once formed, the biologically protected polypeptide is reacted with chemical protecting agents to protect the side chain groups and then is cleaved with a cleavage reagent specific for the biologically added protecting group. By these means, a polypeptide is produced having an unprotected N-terminal amino group and protected side chain reactive groups. The unprotected N-terminal amino group is modified with a modifying agent to form an N-terminally modified and side-chain-protected polypeptide. It is then deprotected to form an N-terminally modified polypeptide. EP 651,761 suggests that any sequence of amino acids may be attached as biologically added protecting groups. However, in mammalian expression systems, EPO is expressed with a leader signal sequence which is cleaved off by a signal peptidase in order to yield the processed, mature EPO. Such signal peptidases recognise only restricted amino acid residues at the P1′ and P3′ cleavage site (R. E. Dalbey et al. Protein Sci. 6, 1129 (1997). Thus, a biologically added protecting peptide has to be built up from an N-terminal amino acid sequence of at least three amino acids for cleavage of the signal sequence, followed by an amino acid sequence for enzymatic or chemical removal of the protecting group. If the recognition sequences of both the signal peptidase and the cleavage protease are identical or closely related, then the sequence of the biologically added protecting group can be reduced to a few amino acids.
In another method, N-terminal selective modification is obtained by chemoselective ligation to an aldehyde (or ketone)-functionalized target macromolecule (European Patent Application EP 788,375; Gaertner, H F, Offord, R E, Bioconjugate Chem., 7 (1), 38–44 (1996)). However, this method only works for N-terminal serines or threonines.
In yet another method, selective modification at N-terminal alanine is accomplished by transamination of alanine to pyruvate (European Patent Applications EP 964,702 and EP 605,963). The disadvantage of this method is that the EPO derivative obtained possessed reduced in vitro activity. Also the transformation agents Cu2+/glyoxylic acid/NaOAc are likely to produce side reactions within the EPO molecule.
Site specific N-terminal modification was also shown by transglutaminase-mediated incorporation of poly(ethylene glycol) derivatives (Sato, H., Yamamoto, K, Hayashi, E, Takahara, Y, Bioconjugate Chem. 11(4), 502–509 (2000)). But this method showed only low yields and needs incorporation of a peptide tag at the N-terminus and hence modifies the polypeptide structure.
Modification with glyoxylyl-based labelling reagents also enables selective N-terminal modification (Zhao, Z G, Im, J S, Clarke, D F, Bioconjugate Chem. 10, 424–430 (1999)) but is restricted to cysteine.
There exists a need for a pegylated EPO composition that is essentially comprised of a single EPO positional isomer and can readily be synthesized with current state of the art production-scale technology.